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Nanoparticle Mediated Mitochondrial Damage Induces Apoptosis in Cancer Abhik Mallick, Piyush More, Muhammed Muazzam Kamil Syed, and Sudipta Basu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b00263 • Publication Date (Web): 10 May 2016 Downloaded from http://pubs.acs.org on May 11, 2016

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ACS Applied Materials & Interfaces

Nanoparticle

Mediated

Mitochondrial

Damage

Induces Apoptosis in Cancer Abhik Mallick, Piyush More, Muhammed Muazzam Kamil Syed, Sudipta Basu* Department of Chemistry, Indian Institute of Science Education and Research (IISER)-Pune, Pune, 411008, Maharashtra, India Corresponding Author *Email: [email protected]

ACS Paragon Plus Environment

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KEYWORDS: Mitochondria, Nanoparticle, Cancer, Obatoclax, α-tocopheryl succinate ABSTRACT: Detouring of conventional DNA damaging anti-cancer drugs into mitochondria to damage mitochondrial DNA is evolving as a promising strategy in chemotherapy. Inhibiting single target in mitochondria would eventually lead to emergence of drug resistance. Moreover targeting mitochondria selectively in cancer cells, keeping them intact in healthy cells, remain a major challenge. Herein, triphenylphosphine (TPP) coated positively charged 131.6 nm spherical nanoparticles (NPs) comprised of α-tocopheryl succinate (TOS, inhibitor of complex II in electron transport chain) and obatoclax (Obt, inhibitor of Bcl-2) were engineered. The TOS-TPPObt-NPs entered into acidic lysosomes via macropinocytosis followed by lysosomal escape and finally homed into mitochondria over a period of 24 h. Subsequently, these TOS-TPP-Obt-NPs triggered mitochondrial outer membrane permeabilization (MOMP) by inhibiting anti-apoptotic Bcl-2, leading to cytochrome c release. These TOS-TPP-Obt-NPs mediated mitochondrial damage induced cellular apoptosis through caspase-9 and caspase-3 cleavage to show improved efficacy in HeLa cells. Moreover, TOS-TPP-Obt-NPs induced MOMP in drug resistant triple negative breast cancer cells (MDA-MB-231) leading to remarkable efficacy compared to the combination of free drugs in higher drug concentrations. Results presented here clearly stimulate the usage of multiple drugs to perturb simultaneously diverse targets, selectively in mitochondria, as next generation cancer therapeutics.


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1. INTRODUCTION In last couple of decades, cancer has emerged as one of the most life threatening diseases globally.1,2 Current chemotherapies inhibiting traditional targets or tyrosine kinases, eventually led to drug resistance through intrinsic or extrinsic mechanisms.3,4 As a result, a huge effort has been dedicated to explore novel targets and biomarkers to improve therapeutic outcomes and overcome drug resistance.5,6 Mitochondrion has recently emerged as one of the major targets in cancer therapy due to their central role in cellular metabolism, sub-cellular signaling hub, key modulator of programmed cell death and being the alternative home of cellular genomic materials.7-9 Moreover, mitochondria are the ubiquitous powerhouses of the healthy as well as cancer cells.10 Hence, selective sub-cellular targeting of mitochondria in cancer cells, keeping them unperturbed in healthy cells, could be of central importance for the development of cancer therapeutics and therefore demanding nanotechnology-based toolkits.11,12 Several nanovectors have been demonstrated to deliver small molecule drugs,13 nucleic acids (genes and siRNAs)14 and therapeutic proteins successfully into tumor tissues through passive and active targeting.15 Targeting of sub-cellular organelles by nano-vectors remain largely unexplored. Dhar et al. developed lipophilic cationic triphenylphosphine (TPP) coated nanoparticles to deliver DNA damaging drug (cisplatin) or metabolic inhibitor (3bromopyruvate) into mitochondria by exploiting their higher membrane potential compared to cell membrane.16-18 Similarly, several other nanovectors were also developed to deliver therapeutic molecules into mitochondria through TPP or cationic peptide or other cationic moiety mediated targeting.19-29 However, strategies for targeting mitochondria by delivering single DNA damaging drug would eventually lead drug resistance through either mitochondrial DNA damage


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repair mechanisms or nuclear cross-talk.30 Recently, we have engineered nanoparticles from direct conjugation of mitochondria damaging and nucleus damaging drugs for simultaneous targeting of both the organelles for improved therapeutic outcome in cancer.25 Interestingly, nanotechnology based platforms have unique advantages to overcome drug resistance by increasing the effective drug uptake and reduce drug ejection through drug efflux pumps.31-33 Furthermore, in the post-genomic era, rational combinatorial drug therapy has emerged as interesting strategy for cancer to maximize therapeutic efficacy and reduce drug resistance.34-37 As a result, there is an urgent need for developing nanotechnology-based mitochondria directed polypharmacology to overcome drug resistance,38 improve the therapeutic efficacy and reduce the toxic side effects for the cancer patients. To address the aforementioned important issues, herein we have engineered nanoparticles (NPs) comprised of triphenylphosphine (TPP), α-tocopheryl succinate (TOS; mitochondrial electron transport chain inhibitor) and obatoclax (Obt; Bcl-2 inhibitor) moieties. These TOS-TPP-ObtNPs were localized in lysosomes by macropinocytosis within 1 h followed by lysosomal escape in 6 h. Finally TOS-TPP-Obt-NPs homed into mitochondria over 24 h. These TOS-TPP-Obt-NPs activated mitochondrial outer membrane permeabilization (MOMP) through anti-apoptotic Bcl-2 inhibition, followed by cytochrome c release. This nanoparticle mediated MOMP lead the cleavage of caspase-9 and caspase-3 to induce apoptosis in cervical cancer cells (HeLa) (Figure 1c). Interestingly, these TOS-TPP-Obt-NPs exhibited improved effects in drug resistant MDAMB-231 cells by damaging mitochondria. These nanoparticles could be used as a platform to deliver multiple drugs into mitochondria of cancer cells to damage different targets simultaneously.


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2. MATERIALS AND METHODS 2.1 Materials. Solvents, α-tocopherol succinate, 5-bromopentanol, 4-(dimethylamino) pyridine (DMAP),

triphenylphosphine, Calcein AM, cobalt(II) chloride (CoCl2), chlorpromazine,

amiloride, genistein, L-α-phosphatidylcholine (PC), sephadex G-25, sodium dodecyl sulfate (SDS), Hanks' balanced solution without phenol red and sodium bicarbonate, 4-(2Hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt (HEPES), acridine orange, lab tek chamber slide 8 wells, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC 1 dye) and silicon wafer for field emission scanning electron microscopy (FE-SEM) were bought from Sigma-Aldrich. Obatoclax mesylate was bought from Selleck Chemicals. Dulbecco’s Modified Eagle’s Medium (DMEM) and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), propidium iodide and

4′,6-Diamidino-2-phenylindole

dihydrochloride (DAPI) were purchased from Gibco. LysoTrackerTM Green DND-153, MitoTracker Green® FM, SlowFade® diamond antifade reagent and were obtained from Life Technologies. Primary antibodies: Anti-Caspase-3 antibody, anti-Bcl-2 antibody, Anti-Caspase9 antibody, Anti-cleaved caspase-3 antibody and Anti tubulin antibody ; Secondary antibodies: HRP Goat anti-rat IgG, HRP Goat anti-mouse IgG, and Annexin V-FITC labeling solution were bought from biolegend. The mitochondria isolation kit for cultured cells was bought from the Thermo Scientific. 2.2 Synthesis of conjugate 2. α-tocopherol succinate (1) (0.2 g, 0.3773 mmol) was activated by N,N'-dicyclohexylcarbodiimide (DCC) (0.093 g, 0.4524 mmol) and DMAP (0.022 mg, 0.1885 mmol). 5-bromopentanol (91.26 µL, 0.754 mmol) was reacted after 30 min and the reaction was continued for 3 h. Filtration was done by Whatman filter paper to remove the excess solid


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dicyclohexylurea (by product). Pure compound 2 was obtained in 50.75 % yield by column chromatography. 2.3 Synthesis of TOS-TPP conjugate (3). Conjugate 2 (0.124 g, 0.1825 mmol), triphenylphosphine (0.095 g, 0.365 mmol) and 70 mg of potassium carbonate (K2CO3) were taken in a round bottom flask, dissolved in 5 mL of acetonitrile (CH3CN) and refluxed for 24 h. After the reaction was over (monitored by TLC), K2CO3 was filtered and rinsed with CH3CN. The filtrate was evaporated using rotary evaporator and then treated with diethylether to remove the excess triphenylphosphine. Pure compound 3 was obtained in 35 % yield after column chromatography. 2.4 Engineering of TOS-TPP-Obt-NPs. PC, α-tocopheryl succinate-TPP conjugate (3), DSPEPEG2000 and obatoclax (Obt) were dissolved in DCM in 1:2:0.2:0.2 weight ratio. DCM was removed thoroughly to form lipid film. Obatoclax containing lipid film was aquated by H2O for 60 min at 60 °C. Self-assembled nanoparticles formed were passed through Sephadex G-25 followed by extrusion through Whatmann membrane having 0.2 µm porosity. 25 2.5 Size, shape, surface charge determination. Size, shape, surface charge, morphology and elemental analysis of the TOS-TPP-Obt-NPs were evaluated by dynamic light scattering (DLS), field-emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDX). The sample preparation and imaging were done according to the reference 25. 2.6 Determination of drug loading. Concentrations versus absorbance graphs were plotted for α-TOS (λmax = 287 nm) and obatoclax (λmax = 488 nm) by UV-Vis spectroscopy. Drug loading in NPs was calculated from the calibration graph in triplicate. Drug loading efficiency and drug loading content were calculated by the equations below.


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Drug loading efficiency = Amount of drug loaded into nanoparticle ÷ Amount of drug used X 100 %. Drug loading content = Amount of drug loaded into nanoparticle ÷ Amount of nanoparticle X 100 %. 2.7 Stability. Stability of the TOS-TPP-Obt-NPs in different conditions was done using the same protocol in reference 25. 2.8 Release kinetics. Release of obatoclax and α-TOS from TOS-TPP-Obt-NPs in pH = 5.5 and pH = 7.4 were performed by using dialysis method described in reference 25. 2.9 In vitro assays. Cellular internalization, cell viability by MTT assay, apoptosis detection by fluorescence activated cell sorting (FACS) and western blot analyses were performed by the procedure described in the reference 25. 2.10 Endocytosis mechanism by CLSM. 5 x 104 HeLa cells were attached on a cover slip for 12 h, followed by washing with PBS. Cells were pre-incubated with following inhibitors: Chlorpromazine (10 µg mL-1), 5-(N-ethyl-N-isopropyl)-Amiloride (1 mM) and Genistein (200 µM) for 30 min. Control cells were treated without inhibitors. After 30 min, the inhibitor solutions were removed, and TOS-TPP-Obt-NPs (2 µg mL-1) in media were added and incubated for 2 h. Cells were fixed with paraformadehyde. Lysosomes were labeled by 1 µM LysoTrackerTM Green DND-153. The nuclei of the cells were further labeled with 2 µg mL-1 DAPI. After three more washings with PBS, cells were imaged by confocal laser scanning microscopy (CLSM) (Zeiss LSM 710). 2.11 Endocytosis mechanism by flow cytometry (FACS). 5 x 104 HeLa cells were cultured and then pre-incubated with chlorpromazine, 5-(N-ethyl-N-isopropyl)-amiloride and genistein in the same concentrations described before for 30 min. After 30 min, the inhibitor solutions were


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removed and TOS-TPP-Obt-NPs were added and incubated for 2 h. A control was kept without adding the nanoparticle. After 2 h, the cells were detached by trypsin and individual cells were analyzed by flow cytometry. 2.12 Lysosome permeabilization by acridine orange assay. 5 x 104 HeLa cells were incubated with TOS-TPP-Obt-NPs (10 µg mL-1) for 12 h. A control was kept without adding the nanoparticle. After PBS wash, cells were treated with acridine orange (5 µg mL-1) for 15 min. Cells were then fixed with paraformadehyde and imaged by CLSM. 2.13 Mitochondria localization. 1.5 x 104 HeLa cells were seeded in a lab tek chamber slide 8 well (per well). After 12 h attachment, cells were incubated with TOS-TPP-Obt-NPs for 12 h and 24 h. Mitochondria were labeled with MitoTracker Green® FM and imaged by CLSM. 2.14 JC1 assay. 1.5 x 104 HeLa cells were seeded in a lab tek chamber slide 8 well (per well). TOS-TPP-Obt-NPs were incubated into the cells for 24 h. A control was kept without adding the nanoparticle. Cells were then washed twice with PBS (pH = 7.4) and incubated with JC1 dye (10 µg mL-1) at 37 °C for 20 min. Before adding JC1 dye, cells were visualized using CLSM at 590 nm fluorescence emission wave length and the red fluorescence signal obtained from the nanoparticles were calculated to be deducted from JC1 fluorescence emission signal as base line correction. The JC1 dyes in cells were visualized and quantified by CLSM. 2.15 Calcein AM assay. 1.5 x 104 HeLa cells were seeded in a lab tek chamber slide 8 well (per well) and TOS-TPP-Obt-NPs were incubated with the cells for 24 h. A control was kept without adding the nanoparticle. Cells were treated with 1 µM Calcein AM and 1 mM CoCl2 in modified Hanks' balanced solution supplemented with 10 mM Na-HEPES (pH = 7.4) without phenol red and sodium bicarbonate. The cells were imaged by CLSM.


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2.16 Drug content in mitochondria fraction by FACS. 3 x 108 HeLa cells were treated with TOS-TPP-Obt-NPs for 24 h. A control was kept without adding the nanoparticle. After 24 h, cells were collected as pellet after trypsin treatment. The mitochondria was isolated from the cell pellet by the mitochondria isolation kit for cultured cells (Thermo Scientific) using the protocol described by the manufacturer. Briefly, protease inhibitor was added into the Reagent A and Reagent C. 800 µL of mitochondria isolation Reagent A was added to the cell pellet and vortexed at a medium speed for 5 sec and the tube was incubated into ice for 2 min. Into it, 800 µL mitochondria isolated Reagent B was added and mixed them by vortexing for 5 sec followed by incubation in ice for 5 min. Again 800 µL of mitochondria isolation Reagent C was added into the mixture and mix thoroughly without vortexing. The tube was centrifuged at 700 Xg for 10 min at 4 °C. The supernatant was transferred into new 2 mL tube and centrifuged at 12000 Xg for 15 min at 4 °C. The supernatant was transferred into new tube (cytosolic fraction) and the pellet contained mitochondria. Finally, 500 µL mitochondria isolation Reagent C was added to the pellets and centrifuged at 12000 Xg for 5 min. The supernatant was discarded and mitochondria pellet was isolated. The drug content in the isolated mitochondrial fraction was measured by BD FACS CaliburTM with 10000 total even collected.

3. RESULTS AND DISCUSSION 3.1 Engineering of TOS-TPP-Obt NPs. α-tocopheryl succinate (α-TOS) damages mitochondria due to its ability to bind with mitochondrial complex II of electron transport chain and induce apoptosis in different types of cancer through mitochondrial outer membrane permeabilization (MOMP).39,40 Also, α-TOS demonstrated enhanced effects with traditional chemotherapeutic drugs.41-43 Recently, we have demonstrated that α-TOS-chemotherapeutic drug conjugated NPs can simultaneously target mitochondria and nucleus in cancer cells.25 In this current study, we


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appended a linker with α-TOS by conjugating 5-bromopentane-1-ol by esterification using DCC/DMAP. This reaction produced compound 2 (yield = 65%) (Figure 1a). We further introduced mitochondria targeting TPP group by reacting compound 2 with triphenylphosphine in presence of potassium carbonate (K2CO3) as base in refluxing condition to afford α-TOS-TPP conjugation product 3 (yield = 34 %).44 We characterized conjugate 2 by 1H,

13

C nuclear

magnetic resonance spectroscopy (NMR), high-resolution mass spectroscopy (HR-MS) and matrix assisted laser adsorption/ionization-time of flight (MALDI-TOF) mass spectroscopy. In 1

H-NMR spectra of conjugate 2, appearance of triplate peaks at δ = 4.11-4.14 ppm (2H) and

3.37-3.41 ppm (2H) confirmed the presence of Br-(CH2)4-CH2-OCO-TOS protons and Br-CH2(CH2)4-OCO-TOS protons respectively (Figure S1). Also multiplate peaks at δ = 1.84-1.91 ppm (2H), 1.74-1.81 ppm (2H) and 1.64-1.69 ppm (2H) confirmed the presence of Br-CH2-(CH2)3CH2-OCO-TOS protons. Moreover, the presence of triplate peaks at δ = 2.92-2.95 ppm (2H), 2.74-2.78 ppm (2H) and 2.57-2.60 ppm (2H) represented Br-(CH2)5-OCO-CH2-CH2-COO-TOS, Br-(CH2)5-OCO-CH2-CH2-COO-TOS and benzylic –CH2- protons on tetrahydropyran moiety in TOS respectively (Figure S1). Finally, the aromatic –CH3 protons were confirmed by singlate peaks δ = 2.09 ppm (3H), 2.02 ppm (3H) and 1.98 ppm (3H) in the TOS moiety (Figure S1). 13C NMR spectra of conjugate 2 revealed characteristic peaks at δ = 172.2 ppm and 170.9 ppm for the ester functionality (a and b peaks in Figure S2). All six aromatic carbon atoms appeared at characteristic δ values of 149.6, 141.3, 126.7, 124.9, 123.0, and 117.4 ppm (d, e, f and g peaks in Figure S2). The quaternary carbon atom n the tetrahydropyran moiety and Br-(CH2)4-CH2-OCOTOS carbon atom in the linker were characterized by the peaks at δ = 75.1 ppm and 64.5 ppm respectively. Finally, conjugate 2 was confirmed by HR-MS and MALDI-TOF spectroscopy by having molecular ion peak [M]+ at m/z = 679.3929 (Figure S3), [M+Na]+ at m/z = 701.3763 and [M+K]+ at m/z = 717.3459 respectively (Figure S4). Conjugate 3 was also characterized by 1H, 13

C NMR spectroscopy, HR-MS and MALDI-TOF mass spectrometry. The appearance of

aromatic peaks as multiplates in 1H NMR spectra of conjugate 3 at δ = 7.67-7.85 ppm (15H) confirmed the presence of phenyl groups (j peaks in Figure S5). The remaining characteristic proton peaks appeared in the respected positions as in conjugate 2 (a, b, c, d, e, f, g, h and i peaks in Figure S5). Similarly, in 13C NMR spectra, new aromatic carbon peaks appeared at δ = 134.9, 133.6, 130.5, 118.8, 117.9 and 117.4 ppm confirmed the presence of phenyl groups in conjugate 3 (F peaks in Figure S6). The other peaks remained in the same position as in conjugate 2 (a, b,


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c, d, e, f, g, h peaks in Figure S6). Conjugate 3 was also characterized by molecular ion peaks [M]+ at m/z = 861.5582 by HR-MS (Figure S7) as well as by MALDI-TOF mass spectrometry (Figure S8). We also characterized the presence of TPP group in conjugate 3 by

31

P NMR

spectroscopy having characteristic peak at δ = 24.3 ppm (Figure S9). We finally evaluated the purity of the conjugates 2 and 3 by reverse phase high-performance liquid chromatography (RPHPLC). Conjugate 2 and conjugate 3 showed 93.4 % and 95.6 % purity respectively in RPHPLC (Figure S10). All these 1H,

13

C,

31

P NMR, HR-MS, MALDI-TOF and RP-HPLC data

clearly confirmed the presence of conjugate 2 and conjugate 3 in high purity. We have engineered mitochondria targeted dual drug loaded TOS-TPP-Obt-NPs by lipid self-assembly in water (Figure 1b).45 We have chosen obatoclax as the second drug to be entrapped into the nanoparticle. Obatoclax is a red fluorescent small molecule, BH3-domain binding pan-Bcl-2 inhibitor, already in a clinical trial as monotherapy as well as in combination with other drugs for different types of cancers.46-48 Moreover, obatoclax is highly hydrophobic in nature leading to erratic biodistribution to bind with its actual target on mitochondrial outer membrane.49 We synthesized TOS-TPP-Obt-NPs by mixing phosphatidylcholine (PC), α-TOS-TPP conjugate 3 and free obatoclax in 2:1:0.2 weight ratios. We used DSPE-PEG to surface coat the nanoparticles with polyethylene glycol to evade the mononuclear phagocytic systems (MPS) to ensure prolonged blood circulation.50 The size of the nanoparticles is one of the important determining factors to be successfully accumulated into the tumor tissues by unique leaky vasculature.51 In a recent report, it was shown that the nanoparticles having optimal diameter range of 100-160 nm had improved blood circulation and tumor accumulation by enhanced permeability and retention (EPR) effect.52 The mean hydrodynamic diameter of the TOS-TPP-Obt-NPs was determined to be 131.6 ± 1.62 nm (mean ± SEM, n = 3) (Figure 2a) by dynamic light scattering (DLS). Hence, we expect our TOS-TPP-Obt-NPs will also have better blood circulation half-life as well as improved tumor tissue homing by EPR effect. For effective mitochondria trafficking, the nanoparticles should bear high positive charge on the surface. We also evaluated the surface charge of the TOS-TPP-Obt-NPs by DLS, which was found to be + 42.9 ± 1.20 mV (mean ± SEM, n = 3) (Figure 2b). Therefore, this highly positively charged sub 200 nm TOS-TPP-ObtNPs should effectively home into tumor tissues followed by mitochondria localization. Size, shape and morphology of the nanoparticles play critical role to be recognized by body’s innate immune system.53 Thus, we visualized the nanoparticles by electron microscopy. The FESEM


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(Figure 2c) and TEM images (Figure 2d) clearly showed that TOS-TPP-Obt-NPs were monodispersed in nature having spherical in shape with smooth surface morphology and less than 200 nm in diameter. The spherical shape and smooth surface morphology were also confirmed by AFM images (Figure S11) with little aggregation in nature. We also determined the presence of nitrogen in obatoclax and phosphorus in triphenylphosphine (TPP) in the same TOS-TPP-ObtNPs by EDX (Figure S12). We evaluated dual drug loading in the nanoparticle by UV-Vis at λmax = 287 nm (α-TOS) and λmax = 488 nm (obatoclax) (Figure S13a-b). The loading of α-TOS and obatoclax was 1035 µg/mL (loading efficiency = 20.7%, loading content = 6.4 %) and 222 µg/mL (loading efficiency = 22.2%, loading content = 1.3%) respectively (Figure S13c). In our previous study, we have observed that α-TOS-drug nanoparticles formed 10 nm hydrophobic shells with hydrophilic core.25 To understand the location of hydrophobic obatoclax molecules in our TOS-TPP-Obt-NP, we evaluated the fluorescence emission spectra of free obatoclax and compared with the fluorescence emission spectra of encapsulated obatoclax in TOS-TPP-Obt-NPs (Figure 2e). It was observed that the characteristic peak of obatoclax at λmax = 546 nm was completely quenched in TOS-TPP-Obt-NP which clearly indicated the π-π stacking of highly aromatic planner obatoclax with the aromatic ring of α-TOS in the hydrophobic region. Moreover, fluorescence steady state anisotropy study showed a 3 folds increase in fluorescent anisotropy of encapsulated obatoclax in TOS-TPP-Obt-NPs compared to free obatoclax (Figure 2f), which conclusively demonstrated that obatoclax resides in the hydrophobic shells in the TOS-TPP-ObtNPs. For successful clinical translation, the TOS-TPP-Obt-NPs should have shelf stability at 4 °C and at 37 °C. The stability of the TOS-TPP-Obt-NPs at 4 °C was assessed through DLS. The diameter was increased from 133.9 ± 0.5 nm to 164.7 ± 2.4 nm at 7 days (Figure S14a). At the same time, the PDI value was also increased from 0.141 ± 0.01 to 0.39 ± 0.007 over a week (Figure S14b). The stability of TOS-TPP-Obt-NPs at 37 °C in phosphate buffer saline (PBS) was also measured. The diameter of the nanoparticles changed from 133.9 ± 0.5 nm to 166.5 ± 1.2 nm over 72 h (Figure S14c). Similarly, PDI value was also increased from 0.141 ± 0.01 to 0.348 ± 0.029 in the same time (Figure S14d). Finally, the stability of these TOS-TPP-Obt-NPs were determined in complete DMEM media (with 10 % FBS) at 37 °C. The diameter of the nanoparticles was changed from 131.6 ± 1.6 nm with PDI = 0.141 ± 0.01 to 163. 6 ± 1.2 nm with


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PDI = 0.392 ± 0.018 over 48 h (Figure S14e-f) which is enough to be accumulated in tumor tissues by passive targeting. These stability data clearly demonstrated that TOS-TPP-Obt-NPs were stable in storage condition for a week and in blood circulation mimic for 2 days.

3.2 Cellular internalization. Despite the development of different nano-vectors to deliver the traditional chemotherapeutic drugs to mitochondria, the actual mechanism of mitochondria homing of those nano-vectors is not very well understood.21, 54 To address this, we first evaluated the cellular uptake mechanism of our TOS-TPP-Obt-NPs in HeLa cervical cancer cells using confocal microscopy. Our previous understanding revealed that α-TOS-drug nanoparticles home into lysosomes over 6 h.25 Inspired by those observations, we stained the acidic lysosomes of HeLa cells by LysoTracker DND-153 (green). We incubated the cells with TOS-TPP-Obt-NPs (red) at different time. Cell nucleus was labeled by blue fluorescent DAPI. The merging of green and red fluorescence leading to yellow color in confocal laser scanning microscopy (CLSM) images (Figure 3) confirmed that the TOS-TPP-Obt-NPs localized into the lysosomes within 1 h. Further quantification from CLSM through Pearson’s coefficient and Mander’s coefficients revealed that the volume of overlapping regions of green and red fluorescence gradually decreased from 84.9 % to 80.7 % to 47.4 % from 1 h to 3 h to 6 h respectively (Table S1). This temporal reduction of colocalization volume unmistakably indicated that the TOS-TPP-Obt-NPs escaped from lysosomal compartments in time dependent manner. Besides, in a high resolution CLSM imaging (Figure S15) revealed green fluorescence regions at 6 h, which clearly confirmed that TOS-TPP-Obt-NPs were no longer present in some of the lysosomes. These CLSM images exhibited that TOS-TPP-Obt-NPs would quickly internalize and localize into lysosomes within 1 h but would escape over a period of time leading to the reduction of co-localization over 6 h. We also incubated the HeLa cells with free obatoclax. The cells then were imaged to visualize the localization of free drug at different time points. Contrast to TOS-TPP-Obt-NPs, free obatoclax internalized into the cells and localized into lysosomes as well as nucleus in a non-specific manner (Figure S16). The overlapping of red and blue colors yielded purple color, which showed the co-localization of free obatoclax in nucleus. To be effective in mitochondria targeting, the TOS-TPP-Obt-NPs should not release their payloads in lysosomal acidic environment. Therefore, we evaluated the liberation of drugs from the NPs at acidic pH, which mimics the lysosomes. We incubated TOS-TPP-Obt-NPs in pH =


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5.5 buffer and quantified the release of obatoclax and α-TOS at different pre-determined time points. The amount of drug released was quantified by UV-Vis spectroscopy at λmax = 287 nm (for α-TOS) and λmax = 488 nm (for obatoclax) (Figure S13a-b). Interestingly, at 6 h, only 3.27 ± 0.43 % obatoclax and 10.03 ± 1.52 % α-TOS were released (Figure S17a). Moreover, at 12 h, only 6.20 ± 1.0 % and 15.78 ± 2.09 % of obatoclax and α-TOS were found to be released respectively. This release kinetics data obviously demonstrated that very less amounts of dual drugs were released in lysosomes even after 12 h, which would lead to deployment of the major amount of drugs into mitochondria. Data presented here were measured to be as mean ± SEM (n = 3).

3.3 Mechanism of endocytosis. The cells can engulf different molecules through myriad of endocytosis mechanisms.55 To understand the endocytosis machinery involved, we pre-incubated the HeLa cells with different endocytosis inhibitors (genistein, chlorpromazine and amiloride), followed by treatment with TOS-TPP-Obt-NPs for 2 h. We have incubated the inhibitor treated cells with TOS-TPP-Obt-NPs for 2 h as we have observed that the highest amount of internalization of nanoparticles occurred within 1 h to 3 h. We evaluated the cellular internalization of TOS-TPP-Obt-NPs by CLSM. Figure 4a demonstrated that chlorpromazine and genistein pre-treated cells endocytosed TOS-TPP-Obt-NPs in the same extent as noninhibitor treated control cells and localized into lysosomes (yellow color formed from overlapping of green and red color). However, amiloride treated cells internalized the TOS-TPPObt-NPs significantly less in comparison with control cells. Pearson’s coefficient and Mander’s coefficients quantification from CLSM images revealed that amiloride treated cells lead to remarkable reduction of volume of overlapping to 26.8% (Table S2). In contrast, genistein, chlorpromazine and control cells showed overlapping volume of 76.4 %, 65.5 % and 68.8 % respectively (Table S2). These confocal microscopy studies unambiguously confirmed that TOSTPP-Obt-NPs penetrated into the cells through macropinocytosis which was inhibited by amiloride pre-treatment. On the other hand, cells showed no effects in genistein and chlorpromazine treatment indicating that caveoline-mediated and clathrin-mediated endocytosis machineries respectively were not operational to internalize TOS-TPP-Obt-NPs in HeLa cell. HeLa cells were further pre-incubated by different endocytosis inhibitors (genistein, chlorpromazine and amiloride) followed by TOS-TPP-Obt-NPs for 2 h and evaluated the number


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of fluorescently labeled cells by fluorescence activated cell sorting (FACS) method (Figure 4b). The FACS data clearly demonstrated that the number of fluorescently labeled cells was significantly reduced in amiloride pre-treated cells, whereas the number of cells was negligibly different in genistein and chlorpromazine treatment in comparison to no-treatment. This FACS data obviously supported the mechanism of endocytosis determined by CLSM images. After accumulating into the acidic lysosomes, the TOS-TPP-Obt-NPs must escape lysosomes before trafficking to mitochondria. To evaluate the lysosomal permeabilization, TOS-TPP-ObtNPs were incubated into HeLa cells for 12 h, followed by acridine orange dye and imaged the cells by CLSM. Acridine orange is a lipophilic amine dye, which shows red color while inside the intact lysosomes in acidic condition. However, after lysosomes are permeabilized, acridine orange releases into the cytosol leading to the change in its color from red to green.56 Posttreatment of HeLa cells with TOS-TPP-Obt-NPs showed much intense increase in green fluorescence than red fluorescence in comparison with control cells (Figure 4c). This acridine orange assay clearly delineated that TOS-TPP-Obt-NPs induced degradation of lysosomal integrity after 12 h.

3.4 Mitochondria localization. After lysosomal escape, the TOS-TPP-Obt-NPs should be trafficked to mitochondria to deliver their payloads. To visualize the localization into mitochondria, TOS-TPP-Obt-NPs were incubated into HeLa cells for two different time points (12 h and 24 h) followed by staining the mitochondria with MitoTracker green dye. We imaged the cells by CLSM. Figure 5a exhibited that the red fluorescent TOS-TPP-Obt-NPs co-localized into green fluorescent-tagged mitochondria leading to merge yellow color in both 12 h and 24 h time points. Pearson’s coefficient and Mander’s coefficients based quantification of volume of co-localization demonstrated 77.5 % and 75.6 % overlapping regions in 12 h and 24 h time points which corroborated that TOS-TPP-Obt-NPs localized into mitochondria over 24 h (Table S3). To further confirm the localization of TOS-TPP-Obt-NPs into mitochondria, nanoparticles were incubated into HeLa cells till 24 h (as substantial amount of nanoparticles accumulated into mitochondria at 24 h) and isolated the mitochondria using mitochondria isolation kit.20 As control we isolated the mitochondria from non-nanoparticle treated HeLa cells. We evaluated the presence of red fluorescent obatoclax into mitochondria by flow cytometry analysis. The FACS


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data in Figure 5b demonstrated remarkable increase in red fluorescent intensity in mitochondria in contrast to control due to the accumulation of TOS-TPP-Obt-NPs into mitochondria (Figure S18). These confocal microscopy and flow cytometry analysis clearly exhibited that TOS-TPPObt-NPs successfully trafficked into mitochondria in time dependent manner over 24 h. After being localized into the mitochondria, the TOS-TPP-Obt-NPs should release their cargoes at pH = 7.4 which mimics the mitochondrial outer membrane.57 Hence, we incubated TOS-TPPObt-NPs at pH = 7.4 buffer and quantified the release of obatoclax (λmax = 488 nm) and α-TOS (λmax = 287 nm) by UV-Vis spectroscopy at different time points. We found that after localization into mitochondria at 12 h, 23.48 ± 1.95 % and 47.91 ± 2.8 % obatoclax and α-TOS were released respectively (Figure S17b). We also observed that at 24 h, substantial amount of obatoclax (35.41± 3.15 %) and α-TOS (59.17 ± 2.32 %) were released from the nanoparticles into mitochondria. Furthermore, highest amount of obatoclax (41.25 ± 1.9 %) and α-TOS (69.38 ± 3.4%) were released from TOS-TPP-Obt-NPs at pH = 7.4 at 48 h. This release experiment evidently showed that significantly high amount of dual drugs were released after localization of TOS-TPP-Obt-NPs into mitochondria at 12 h, 24 h and 48 h, which would lead to the effective inhibition of multiple mitochondrial targets. All the data presented here were determined to be as mean ± SEM (n = 3).

3.5 Mitochondrial outer membrane permeabilization. After trafficking into mitochondria and successfully released their dual drugs, we further investigated the effect of TOS-TPP-Obt-NPs on mitochondrial membrane potential (∆ψm) using a cationic dye 5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolylcarbocyanine iodide (JC1). JC1 shows potential reliant accumulation in the mitochondria with fluorescent emission alteration from green (~ 525 nm) to red (~ 590 nm) due to formation of red fluorescent J-aggregates. Mitochondrial depolarization can be measured by the increase in the ratio of green/red fluorescent intensity.58 After 24 h incubation with TOSTPP-Obt-NPs, followed by staining with JC1, the live HeLa cells were visualized by confocal microscopy. Figure 6a revealed significant increase in green/red ratio (green: red = 3.44 ± 0.06) in TOS-TPP-Obt-NP treated cells compared to much less green/red ratio (green: red = 1.27 ± 0.01) in the non-nanoparticle treated control cells as well as only free obatoclax treated cells (green: red = 1.89 ± 0.08) (Figure S19). This JC1 assay exhibited that TOS-TPP-Obt-NPs


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induced mitochondrial depolarization much significantly (2 times more) compared to free obatoclax. Mitochondrial depolarization results the opening of mitochondrial permeability transition pores (MTP). We further evaluated the opening of MTPs by Calcein O,Oˊ-diacetate tetrakis (acetoxymethyl) ester (Calcein AM) dye.59 Calcein AM passively diffuses into the cells and accumulates into cytosol and mitochondria. Inside cells, esterases cleave the ester functionality leading to the release of green fluorescent calcein dye, which is quenched by addition of CoCl2, whereas mitochondrial Calcein AM remains intact. After opening of MTPs, mitochondrial Calcein AM will be released into cytosol leading to conversion into fluorescent Calcein. 24 h post-incubation of cells with TOS-TPP-Obt-NPs, followed by treatment with calcein AM and CoCl2, the live HeLa cells were visualized by confocal microscopy. On the other hand, only Calcein AM and CoCl2 were added into the HeLa cells without any nanoparticles. Confocal microscopy pictures in Figure 6b clearly confirmed the TOS-TPP-Obt-NPs induced opening of mitochondrial transition pores leading to increase in green fluorescence intensity in contrast to no-nanoparticle treated cells. Hence Calcein AM assay demonstrated that TOS-TPP-Obt-NPs induced permeabilization of mitochondrial outer membrane (MOMP). Mitochondrial outer membrane permeabilization leads to the release of cytochrome c from inter membrane space (IMS).60 To visualize the cytochrome c release after mitochondrial damage, TOS-TPP-Obt-NPs were incubated into HeLa for 24 h. The cells were then stained with cytochrome c antibody tagged with green fluorescent dye Alexa Fluor 488 and imaged through CLSM. Figure 6c explicitly exhibited significant amplification in expression of cytochrome c for TOS-TPP-Obt-NPs treatment in contrast to control cells. Hence, these experiments confirmed that after localization into mitochondria and releasing dual drugs after 24 h, TOS-TPP-Obt-NPs triggered mitochondrial depolarization, followed by opening of mitochondrial transition pores leading to release of cytochrome c as pro-apoptotic molecule.

3.6 Cytotoxicity, apoptosis and mechanism. Mitochondrial outer membrane permeabilization (MOMP) and release of cytochrome c as pro-apoptotic molecule induce apoptosis leading to the cellular death.60 To understand the effect of nanoparticle mediated mitochondrial damage in cancer cells, we treated HeLa cells by TOS-TPP-Obt-NPs for 24 h and 48 h followed by evaluating the cell viability by MTT assay. A combination of α-TOS and obatoclax was used to


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treat HeLa cells also. At 24 h, TOS-TPP-Obt-NPs demonstrated much lower IC50 = 6.30 ± 0.22 µM (cell viability = 40.4 ± 2.7 % at 25 µM concentration of obatoclax) compared to IC50 = 13.09 ± 0.48 µM (cell viability = 42.37 ±1.54 % at 25 µM concentration of obatoclax) for free drug cocktail (Figure S20a). Also, at 48 h, TOS-TPP-Obt-NPs showed even more improved efficacy of IC50 = 3.7 ± 0.07 µM compared to IC50 = 6.7 ± 0.1 µM for free drugs combination (Figure 7a). Moreover, TOS-TPP-Obt-NPs induced more cancer cell death (cell viability = 11% at 25 µM concentration of obatoclax) compared to free drug combination in the same α-TOS/obatoclax ratio (cell viability = 14% at 25 µM concentration of obatoclax). We rationalized that TOS-TPPObt-NPs showed much improved effect in cell death at 48 h compared to 24 h due to their improved accumulation in mitochondria at 48 h as well as the enhanced release of dual drugs at 48 h at pH = 7.4 compared to 24 h. In addition, at 24 h, the nanoparticles started to accumulate into the mitochondria which would take another 24 h to release considerable amount of drugs into mitochondria for enhanced efficacy at 48 h (Figure S17b). We also evaluated the effects of free α-TOS, free α-TOS-TPP and free obatoclax in HeLa cells by MTT assay after 48 h of incubation. Free α-TOS, free α-TOS-TPP and free obatoclax illustrated much higher IC50 = 25.26 ± 2.96 µM, 33.97 ± 0.87 µM and 13.38 ±0.2 µM respectively (Figure S20b-d). These cell viability data clearly delineated that dual drug loaded TOS-TPP-Obt-NPs were much more efficacious compared to free single drug treatments as well as drug combinations. To investigate the mechanism triggering HeLa cell death, we evaluated the induction of apoptosis by TOS-TPP-Obt-NPs by FACS analysis. TOS-TPP-Obt-NPs were incubated into HeLa cells for 24 h followed by staining the peripheral phosphatidylserine in cells triggered to apoptosis by green fluorescent Annexin V-FITC. We distinguished cells with necrosis properties from the cells triggered for apoptosis by red fluorescent propidium iodide (PI). Undeniably, TOS-TPP-Obt-NPs induced early apoptosis in 61.3 % cells and late apoptosis in 17.4 % cells. In contrast, only 1.8 % and 0.1 % cells went into early and late apoptosis in control cells. (Figure 7b). Hence, it was evident that TOS-TPP-Obt-NPs induced early apoptosis leading to the cell death in HeLa cells. Obatoclax inhibits anti-apoptotic Bcl-2 in its BH3 domain, triggers the polymerization of BAKBAX leading to MOMP.47 To evaluate the effect of nanoparticles on Bcl-2, TOS-TPP-Obt-NPs were incubated into HeLa cells for 24 h and estimated the level Bcl-2 by gel electrophoresis. Figure 7c demonstrated that TOS-TPP-Obt-NPs successfully inhibited Bcl-2 expression (2.2


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folds reduction, Figure S21a) on mitochondrial outer membrane to induce MOMP. Activating MOMP through Bcl-2 inhibition, followed by cytochrome c release triggers initiator caspase-9 which upon cleavage activates executioner caspase-3 leading to cellular apoptosis through intrinsic mechanism.61,62 To evaluate the effect of MOMP in apoptosis, TOS-TPP-Obt-NPs were incubated into HeLa cells for 24 h and evaluated the expression of pro-caspase-9, cleaved caspase-3 and pro-caspase-3 by western blot analysis. Fig. 7c, showed the reduction of expression of pro-caspase-9 (2.1 folds, Figure S21b) and pro-caspase-3 (1.9 folds, Figure S21d) in TOS-TPP-Obt-NPs treatment in contrast to control. On the other hand, the expression of cleaved caspase-3 was increased (1.9 folds, Figure S21c) in TOS-TPP-Obt-NPs treated cells compared to control cells (Figure 7c). These western blot studies clearly showed that TOS-TPPObt-NPs inhibited Bcl-2 on mitochondrial outer membrane leading to apoptosis through caspase9 and caspase-3 mediated intrinsic mechanism.

3.7 Mitochondria targeting and cytotoxicity in drug resistant cancer cell. One major advantage to target mitochondrial proteins and genomic materials by conventional chemotherapeutic drugs is to overcome the emergence of drug resistance.

16,23,24

Hence, we

evaluated the effect of TOS-TPP-Obt-NPs in drug resistant MDA-MB-231 triple negative breast cancer cells. To visualize the co-localization of TOS-TPP-Obt-NPs in mitochondria, we treated MDA-MB-231 cells with the nanoparticles for 12 h and 24 h time points, followed by staining the mitochondria with MitoTracker green dye and imaged the cells by CLSM. The emergence of yellow color by overlapping green and red fluorescent in Figure 8a explicitly demonstrated that TOS-TPP-Obt-NPs homed into mitochondria over 24 h. Furthermore, quantification of percent volume co-localization revealed that 13.55 % (at 12 h) and 59.49 % (at 24 h) co-localization of TOS-TPP-Obt-NPs took place in mitochondria. (Table S4). To assess the effect of TOS-TPP-Obt-NPs in mitochondrial membrane potential, we treated the MDA-MB-231 cells with the nanoparticles for 24 h and stained the cells with JC1 dye. The live cells were visualized by confocal microscopy. Figure 8b confirmed substantial increase in green/red ratio (green: red = 2.95 ± 0.07) in TOS-TPP-Obt-NP treated cells compared to much decreased green/red ratio (green: red = 0.85 ± 0.04) in the non-nanoparticle treated control cells as well as only free obatoclax treated cells (green: red = 0.58 ± 0.04) (Figure S22).


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Finally, we evaluated the efficacy of TOS-TPP-Obt-NPs in drug resistant triple negative breast cancer MDA-MB-231 cells. We treated the cells with TOS-TPP-Obt-NPs at different concentrations at 48 h and quantified the cell death. TOS-TPP-Obt-NPs showed IC50 = 1.06 ± 0.06 µM whereas free drug cocktail showed very comparable IC50 = 0.78 ± 0.17 µM (Figure 8c). Furthermore, TOS-TPP-Obt-NPs induced much less cell viability (3.2 ± 1.8 % in 25 µM concentration of obatoclax) compared to the cell viability (35.4 ± 7.0 % at 25 µM concentration of obatoclax) induced by the free drug cocktails. More interestingly, TOS-TPP-Obt-NPs showed much improved efficacy in drug resistance triple negative breast cancer MDA-MB-231 cells (IC50 = 1.06 ± 0.06 µM) compared to drug sensitive cervical cancer HeLa cells (IC50 = 3.7 ± 0.07 µM) (Figure S23) at 48 h. We anticipate that our TOS-TPP-Obt-NPs would target Bcl-2 in mitochondria very specifically in cancer cells. It is well know that, Bcl-2 can be localized into mitochondrial outer membrane, nuclear envelop and endoplasmic reticulum to have diverse functions.63 Our novel TOS-TPP-Obt-NPs showed advantage over free drugs (Figure S16) to target specifically mitochondrial Bcl-2 without inhibiting them in other sub-cellular compartments. This nanoparticles mediated spatial targeting of Bcl-2 in mitochondria would reveal interesting properties and behavior in cancer cells. We also envision that, TOS-TPP-ObtNPs would show much improved efficacy in in vivo models to increase the effective drug loading inside the tumor tissue hence increasing maximum tolerated dose (MTD), whereas free drug combination at higher concentration would lead off-target systemic toxicity.45 Finally from these cytotoxicity and apoptosis assays, we can evidently observe that TOS-TPP-Obt-NPs showed much-improved efficacy in cervical cancer cells as well as drug-resistant triple negative breast cancer cells compared to free drug combination through the induction of apoptosis.

4. CONCLUSION In conclusion, we have developed a novel dual-drug loaded nanoparticle entity with positive surface charge for specific targeting of mitochondria in cancer cell. The so called spherical TOSTPP-Obt-NPs are expected to be accumulated into the vascular tumor by enhanced permeability and retention effect. These NPs were found to be endocytosed into the cancer cells by macropinocytosis followed by localization into mitochondria in time dependent manner. In


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mitochondria, TOS-TPP-Obt-NPs triggered mitochondrial outer membrane permeabilization (MOMP) by inhibiting anti-apoptotic Bcl-2. This nanoparticle targeted mitochondrial damage lead to higher cancer cell death through caspase-9 and caspase-3 mediated apoptosis. We envision that the here presented unique approach can be converted from bench to bedside to inhibit numerous diverse targets concurrently into mitochondria, improve the therapeutic efficacy, reduce the off-target toxicity, surmount drug resistance and finally, improve next generation cancer therapy. ASSOCIATED CONTENT Supporting Information. Structural determination by 1H, 13C, 31P NMR spectroscopy, HR-MS, MALDI-TOF, AFM, CLSM details. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Email: [email protected] ACKNOWLEDGMENT S.B.

thanks

DBT

(Ramalingaswami

fellowship:

BT/RLF/Re-entry/13/2011

and

BT/PR9918/NNT/28/692/2013) and SERB (SB/FT/CS-044/2012) for financial support. We sincerely thank Dr. Nirmalya Ballav, IISER Pune for insightful scientific suggestions and manuscript preparation. We also thank DST Nanoscience unit and DST-FIST for providing FESEM and AFM facilities, respectively. We sincerely thank Dr. Mayurika Lahiri from IISERPune and Dr. Manas Kumar Santra from NCCS, Pune for helping in western blot analysis. A.M. is thankful to CSIR-UGC for doctoral fellowship and M.M.K.S is thankful to DST-INSPIRE fellowship for BS-MS degree.


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Figure 1. (a) Synthetic scheme of TOS-TPP conjugate (3) from α-tocopheryl succinate (α-TOS). (b) Engineering of mitochondria targeting TOS-TPP-Obt-NPs. (c) Schematic representation of cellular internalization and mitochondrial homing of TOS-TPP-Obt-NPs leading to apoptosis.


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Figure 2 (a) Hydrodynamic diameter and (b) zeta potential of TOS-TPP-Obt-NPs determined by DLS. All the values were determined as mean ± SEM (n = 3). (c) FESEM (inset scale bar = 200 nm) and (d) TEM (inset scale bar = 500 nm) images of TOS-TPP-Obt-NPs. (e) Fluorescence emission quenching of obatoclax in TOS-TPP-Obt-NPs compared to free obatoclax at λmax = 546 nm. (f) Steady state fluorescence anisotropy of free obatoclax and obatoclax encapsulated in TOS-TPP-Obt-NPs.


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Figure 3. Confocal laser scanning microscopy images of internalization of TOS-TPP-Obt-NPs in HeLa cells in 1 h, 3 h and 6 h time points. Nucleus and lysosomes were stained by DAPI (blue) and LysoTracker (green) dyes. The yellow regions in merged images showed the co-localization of TOS-TPP-Obt-NPs (red) into lysosomes. Scale bar = 10 µm.


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Figure 4. (a) Confocal laser scanning microscopy images of HeLa cells pre-treated with chlorpromazine, genistein and amiloride followed by treatment with TOS-TPP-Obt-NPs. Nucleus and lysosomes were stained by DAPI (blue) and LysoTracker (green) dyes. Scale bar = 10 µm. (b) Fluorescence activated cell sorting analysis of HeLa cells pre-treated with chlorpromazine, genistein and amiloride followed by treatment with TOS-TPP-Obt-NPs. (c) Acridine orange assay in HeLa cells after treatment with TOS-TPP-Obt-NPs which showed much increased green/red ratio compared to non-nanoparticle treated control cells. Scale bar = 10 µm.


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Figure 5. (a) Confocal microscopy images of HeLa cells showing the homing of TOS-TPP-ObtNPs into mitochondria for 12 h and 24 h time points. Mitochondria were stained with MitoTracker green dyes. The yellow regions in the merged images indicate the co-localization of TOS-TPP-Obt-NPs in mitochondria. Scale bar = 10 µm. (b) Flow cytometry analysis of mitochondria fraction isolated from HeLa cells after treatment with TOS-TPP-Obt-NPs for 24 h showing the localization of red fluorescent obatoclax into mitochondria.


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Figure 6. (a) Confocal images of HeLa cells treated with TOS-TPP-Obt-NPs followed by JC1 dye to observe mitochondrial depolarization compared to control cells. The green and red colors were generated by JC1 dye in monomeric and aggregated forms respectively. (b) Confocal images of HeLa cells stained with green fluorescent calcein AM for mitochondrial transition pore (MTP) opening induced by TOS-TPP-Obt-NPs. (c) CLSM images of HeLa cells treated with TOS-TPP-Obt-NPs for 24 h. Cells were stained with cytochrome c Alexa Fluor 488 antibody (green). Scale bar = 10 µm.


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Figure 7. (a) Cell viability assay of TOS-TPP-Obt-NPs in HeLa cells after 48 h incubation respectively (p < 0.001, Two-way ANOVA test). (b) Flow cytometric analysis of apoptosis induced by TOS-TPP-Obt-NPs in HeLa cells. Lower left, lower right, upper right and upper left quadrants represent the % of healthy, early apoptotic, late apoptotic and necrotic cells after treatment with TOS-TPP-Obt-NPs for 24 h. (c) Western blot analysis to visualize the expression of Bcl-2, caspase-9, cleaved caspase-3 and caspase-3 after treatment of HeLa cells with TOSTPP-Obt-NPs for 24 h.


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Figure 8. (a) Confocal microscopy images of MDA-MB-231 cells showing the homing of TOSTPP-Obt-NPs into mitochondria for 12 h and 24 h time points. Mitochondria were stained with MitoTracker green dyes. The yellow regions in the merged images indicate the co-localization of TOS-TPP-Obt-NPs in mitochondria. (b) Confocal images of MDA-MB-231 cells treated with TOS-TPP-Obt-NPs followed by JC1 dye to observe mitochondrial depolarization compared to control cells. The green and red colors were generated by JC1 dye in monomeric and aggregated forms respectively. (c) Cell viability assay of TOS-TPP-Obt-NPs in MDA-MB-231 cells after 48h incubation respectively (p < 0.001, Two-way ANOVA test).


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